Monoclonal Antibodies Against Activated Protein C

ABSTRACT

The present invention provides monoclonal antibodies that selectively bind to and inhibit activated protein C without binding to or inhibiting unactivated protein C. Other antibodies inhibit both activated protein C and activation of unactivated protein C. Methods of treatment employing these antibodies are described herein as are methods of screening for and detecting these antibodies.

BACKGROUND OF THE INVENTION

The present invention claims benefit of priority to U.S. ProvisionalApplication Ser. No. 60/983,092, filed Oct. 26, 2008, the entirecontents of which are hereby incorporated by reference.

1. FIELD OF THE INVENTION

The present invention relates generally to the field of antibodies. Moreparticularly, the present invention describes identification and use ofmonoclonal antibodies and antibody fragments selectively directed toactivated protein C (APC).

2. DESCRIPTION OF RELATED ART

Blood coagulation is a process consisting of a complex interaction ofvarious blood components, or factors, which eventually give rise to afibrin clot. Generally, blood components participating in thecoagulation “cascade” are proenzymes or zymogens—enzymatically inactiveproteins that are converted into an active form by action of anactivator. Regulation of blood coagulation is largely accomplishedenzymatically by proteolytic inactivation of the pro-coagulation factorsVa and VIIIa achieved by activated protein C (APC) (Esmon, 1989).

Protein C is the precursor to APC, a potent natural anticoagulant.Protein C is activated by thrombin in complex with thrombomodulin (TM).The activation is augmented by endothelial cell protein C receptor(EPCR). TM and EPCR can be down-regulated due to inflammatory mediators,such as tumor necrosis factor, reviewed by Esmon (1999). TM and EPCRhave also been found to be reduced in some forms of septic shock,meningococcemia in particular. Since EPCR and TM are expressed onendothelium, it is not possible to directly determine how well they arefunctioning without removal of blood vessels.

APC functions as an anticoagulant by proteolytically cleaving anddownregulating pro-coagulant factors. APC also serves importantfunctions as an anti-apoptosis agent, an anti-inflammatory molecule anda cytoprotectant. Bleeding disorders where homeostatis is dysregulatedthrough a loss of a key factor, such as the absence of Factor VIII inheomphilia, or in trauma patients where the wound process results in atemporary loss of hemostasis, may be treated by the removal of APC. Suchtreatment, however, could result in unwanted detrimental consequences ofremoving the beneficial functions of APC in addition to the removal ofthe anti-coagulant activity. Therefore it is desirable to have atherapeutic that selectively targets the anti-coagulant activity of APCwhile leaving other functions of the molecule intact.

SUMMARY OF THE INVENTION

A method for treating bleeding disorders has been developed anddescribed herein involving the use of a monoclonal antibody whichrecognizes activated protein C, but does not recognized unactivatedprotein C. In this regard, the present invention also providesmonoclonal antibodies that selectively bind to and/or block theproteolytic active site of activated protein C. Such antibodies mayinhibit the anticoagulant activity of activated protein C, but may notaffect any activity of unactivated protein C, in certain embodiments.Such antibodies may also retain the cytoprotective effects of activatedprotein C, in certain embodiments. Thus, methods of the presentinvention also include treatments employing such monoclonal antibodieswhere it is desirable to selectively inhibit the anticoagulant activityof activated protein C.

Accordingly, certain general aspects of the present inventioncontemplate a monoclonal antibody, wherein said antibody binds to andinhibits activated protein C, but does not bind to or inhibitunactivated protein C. For example, certain embodiments of the presentinvention contemplate a monoclonal antibody, wherein said antibody bindsto and inhibits activated protein C anticoagulant activity, but does notbind to or inhibit activation of unactivated protein C. In particularembodiments, a monoclonal antibody of the present invention is HAPC1573. Antibodies of the present invention that bind to and inhibitactivated protein C and its anti-coagulant activities may do so in vivoand/or in vitro.

Other monoclonal antibodies of the present invention are contemplated,such as an antibody that inhibits activated or unactivated protein Cbinding of endothelial cell protein C receptor (EPCR) or phospholipidsand inhibits unactivated protein C activation. In certain aspects, suchan antibody binds to the mouse unactivated protein C Gla-domain. Suchantibodies may be employed in in vitro or in vivo contexts.

An antibody of the present invention may, for example, be a murineantibody, An antibody of the present invention may, for example, be ahumanized antibody. An antibody of the present invention may becomprised in a pharmaceutical composition, wherein the pharmaceuticalcomposition also comprises pharmaceutically acceptable carrier. Anantibody of the present invention may also be used in methods whereinthe antibody is contacted with a cell in vitro or in vivo.

Also contemplated by the present invention is a method of inhibitingactivated protein C anticoagulant activity in a subject, comprisingadministering an effective amount of an antibody of the presentinvention to said subject (e.g., a mammal, such as a human). In this orany other method of the present invention involving the administrationof an antibody of the present invention, the cytoprotective effects ofactivated protein C may, in certain embodiments, not be decreased, ormay stay within normal levels.

Methods of inhibiting activated protein C amidolytic activity in asubject, comprising administering an effective amount of an antibody ofthe present invention to said subject, are also contemplated.

Also contemplated by the present invention is a method of treating asubject in need of blood coagulation comprising administering aneffective amount of an antibody of the present invention to saidsubject. Such a subject could be suffering from, for example, hemophiliaor hemorrhage.

A method of treating a subject suffering from sepsis comprisingadministrating an effective amount of an antibody of the presentinvention is also contemplated herein. Such methods may also employadministration of activated protein C.

Also contemplated are methods of treating a subject suffering fromhemophilia comprising administrating an effective amount of an antibodyof the present invention.

Antibodies of the present invention may also be employed, for example,in methods of modulating hemostasis in a subject or modulatingthrombosis in a subject, comprising administrating an effective amountof an antibody of the present invention. Such methods may also employadministration of activated protein C.

Certain methods of the present invention contemplate a method ofinhibiting activation of unactivated protein C activation comprisingadministering to a subject an effective amount of the monoclonalantibody of the present invention. Antibodies employed in such methodsmay also inhibit activated or unactivated protein C binding ofendothelial cell protein C receptor (EPCR) or phospholipids.

A subject in accordance with present invention may, for example, be amammal, such as a mouse, rat, rabbit, dog, horse, or human.

Unless otherwise noted, any antibody described herein may be an antibodyfragment. For example, the antibody may be further defined as Fab′, Fab,F(ab′)₂, a single domain antibody, Fv, or scFv, which are all well-knowntypes of antibody fragments. Unless otherwise noted, an antibody of thepresent invention also contemplates such fragments.

The term “antibody” is used to refer to any antibody like molecule thathas an antigen binding region, and includes antibody fragments such asFab′, Fab, F(ab′)2, single domain antibodies (DABs), Fv, scFv (singlechain Fv), and the like, described below. The techniques for preparingand using various antibody based constructs and fragments are well knownin the art. Means for preparing and characterizing antibodies are alsowell known in the art (see, e.g., Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988; incorporated herein by reference).

Another aspect of the invention contemplates the variable region thatcomprises alternating complementarity determining regions, or CDRs, andframework regions, or FRs. The CDRs are the sequences within thevariable region that generally confer antigen specificity.

The invention also encompasses portions of antibodies that comprisesufficient variable region sequence to confer antigen binding. Portionsof antibodies include, but are not limited to Fab, Fab′, F(ab′)₂, Fv,SFv, scFv (single-chain Fv), whether produced by proteolytic cleavage ofintact antibodies, such as papain or pepsin cleavage, or by recombinantmethods, in which the cDNAs for the intact heavy and light chains aremanipulated to produce fragments of the heavy and light chains, eitherseparately, or as part of the same polypeptide.

mAbs within the scope of the invention also include sequencescorresponding to human antibodies, animal antibodies, and combinationsthereof. The term “chimeric antibody,” as used herein, includesantibodies that have variable regions derived from an animal antibody,such as a rat or mouse antibody, fused to another molecule, for example,the constant domains derived from a human antibody. One type of chimericantibodies, “humanized antibodies,” have had the variable regionsaltered (through mutagenesis or CDR grafting) to match (as much aspossible) the known sequence of human variable regions. CDR graftinginvolves grafting the CDRs from an antibody with desired specificityonto the FRs of a human antibody, thereby replacing much of thenon-human sequence with human sequence. Humanized antibodies, therefore,more closely match (in amino acid sequence) the sequence of known humanantibodies. By humanizing mouse monoclonal antibodies, the severity ofthe human anti-mouse antibody, or HAMA, response is diminished. Theinvention further includes fully human antibodies which would avoid, asmuch a possible, the HAMA response. Production of humanized antibodiesis described in more detail below.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Except insofar asany conventional carrier is incompatible with the active ingredient, itsuse in the therapeutic or pharmaceutical compositions is contemplated.

The term “contact,” when applied to a cell, is used herein to describethe process by which a compound of the invention is delivered to atarget cell or is placed in direct juxtaposition with the target cell.

The term “effective,” as that term is used in the specification and/orclaims (e.g., “an effective amount,” means adequate to accomplish adesired, expected, or intended result.

The term “substantially” and its variations are defined as being largelybut not necessarily wholly what is specified as understood by one ofordinary skill in the art, and in one non-limiting embodimentsubstantially refers to ranges within 10%, within 5%, within 1%, orwithin 0.5%.

The terms “inhibiting,” “reducing,” or “prevention,” or any variation ofthese terms, when used in the claims and/or the specification includesany measurable decrease or complete inhibition to achieve a desiredresult. For example, there may be a decrease of 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,99%, or more, or any range derivable therein, reduction of activitycompared to normal.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device or themethod being employed to determine the value, or the variation thatexists among the study subjects. For example, “about” can be within 10%,preferably within 5%, more preferably within 1%, and most preferablywithin 0.5%.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any compound, method, or compositionof the invention, and vice versa.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. APC ELISA standard curve.

FIG. 2. HAP1573 enhances APC binding on endothelium.

FIG. 3. HAPC1573 facilitates APC internalization into EA cells.

FIG. 4. HAPC1573 alters APC amidolytic activity toward a chromogenicsubstrate.

FIG. 5. HAPC1573 blocks APC anticoagulant activity in a plasma clottingassay.

FIG. 6. HAPC1573 enhances APC cleaving histones.

FIG. 7. Effect of HAPC1573 on APC cytoprotection against histones.

FIGS. 8A-C. MPC1609 and MAPC1591 inhibit APC anti-coagulant activity.(FIG. 8A) bEnd3 cells were incubated with 100 nM FL-APC in the absenceor presence of 125 nM MPC1609 or MAPC1591 for 15 min on ice andsubjected to flow cytometry. (FIG. 8B) bEnd3 cells were incubated with100 nM protein C and 5 nM thrombin in the absence or presence of 100 nMMPC 1609 or MAPC1591 for 15 min at 37° C. and APC activity was measuredby PCa chromogenic substrate. (FIG. 8C) One stage plasma clotting timewas measured with 200 ng/ml in the absence or presence of 5 μg/mlMPC1609 or MAPC1591. Protein C activation and clotting assays wereperformed in duplicate and all errors were within 5%.

FIG. 9. MPC1609 but not MAPC1591 exacerbated mice into lethality withsublethal dose of LPS. BL6 mice were injected intravenously with 10mg/kg LPS with 10 mg/kg MPC1609 or MAPC1591 and survival rates wereindicated.

FIGS. 10A-C. Body temperature, serum IL-6, BUN and creatinine levels ofmice challenged with LPS and MPC1609 or MAPC1591. BL6 mice (4 mice foreach group) were injected intravenously with saline, 10 mg/kg LPS, or 10mg/kg LPS with 10 mg/kg MPC1609 or MAPC1591. (FIG. 10A) Mouse bodytemperature, (FIG. 10B) serum IL-6, (FIG. 10C) serum BUN and creatininelevels were measured 3 or 18 hrs after mice were challenged.

FIGS. 11A-C. MAPC1591 enhances APC cleaving histones. (FIG. 11A) 100ug/ml calf thymus histone H3(left panel) or H4 (right panel) in Opti-MEMwas incubated with or without 100 nM APC in the absence or presence of200 nM MAPC1591 for 1 hr at 37° C. Samples were then subjected toSDS-PAGE and Commassie blue staining. (FIG. 11B) EA.hy926 cells werecultured with calf thymus histones (50 μg/ml) in the absence or presenceof APC (100 nM) and MAPC1591 (200 nM) for 1 hr at 37° C. Cell death wasmeasured by flow cytometry for PI (FL3) positive staining. (FIG. 11C)BL6 mice were injected intravenously with saline, 10 mg/kg LPS, or 10mg/kg LPS with 10 mg/kg MAPC1591 or MPC1609. Plasma samples were taken18 hrs post challenge and subjected to SDS-PAGE and Western blottingusing goat anti-histone H3 antibody.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention relates to the discovery of monoclonal antibodiesthat selectively bind to activated protein C, but not unactivatedprotein C, and specifically inhibit the anti-coagulation activity ofactivated protein C. These and other aspects of the invention aredescribed in greater detail below.

A. Antibody Structure

Antibodies comprise a large family of glycoproteins with commonstructural features. An antibody is comprised of four polypeptides thatform a three dimensional structure which resembles the letter Y.Typically, an antibody is comprised of two different polypeptides, theheavy chain and the light chain. An antibody molecule is comprised ofone or more Y-units, each Y comprising two heavy chains and two lightchains.

An antibody molecule typically consists of three functional domains: theFc, Fab, and antigen-binding site. The Fc domain is located at the baseof the Y. The arms of the Y comprise the Fab domains. Theantigen-binding site is located at the end of each arm of the Y. Thearea at the fulcrum of the arms of the Y is the hinge region.

There are five different types of heavy chain polypeptides designated asα, δ, ε, γ, and μ. There are two different types of light chainpolypeptides designated κ and λ. An antibody typically contains only onetype of heavy chain and only one type of light chain, although any lightchain can associate with any heavy chain.

The carboxyl terminal of each heavy chain polypeptide is known as theconstant (Fc) region. The amino terminal of each heavy and light chainpolypeptide is known as the variable (V) region. Within the variableregions of the chains are hypervariable regions known as complementaritydetermining regions (CDRs). The variable regions of one heavy chain andone light chain associate to form an antigen-binding site. Each heavychain and each light chain includes three CDRs. The six CDRs of anantigen-binding site define the amino acid residues that form the actualbinding site for the antigen. CDR variability accounts for the diversityof antigen recognition.

B. Preparation of Monoclonal Antibodies of the Present Invention

The present invention concerns the production and use of molecules thatare capable of “specific binding” to one another. As used herein, amolecule is said to be capable of “specific binding” to anothermolecule, if such binding is dependent upon the respective structures ofthe molecules. The known capacity of an antibody to bind to an immunogenis an example of “specific binding.” Such interactions are in contrastto non-specific binding that involve classes of compounds, irrespectiveof their chemical structure (such as the binding of proteins tonitrocellulose, etc.) Most preferably, an antibody of the presentinvention will exhibit “highly specific binding,” such that they will beincapable or substantially incapable of binding to closely relatedheterologous molecules. Indeed, the preferred monoclonal antibodies ofthe present invention exhibit the capacity to bind to activated proteinC, but are substantially incapable of binding unactivated protein C. Infurther embodiments, the monoclonal antibodies specifically inhibit onlythe anticoagulant activities of APC by binding to and blocking theproteolytic active site of APC.

Thus, in one embodiment, such molecules will comprise fragments (such as(F(ab′), F(ab′)2) that are produced, for example, by the proteolyticcleavage of the mAbs, or single-chain immunoglobulins producible, forexample, via recombinant means. Such antibody derivatives aremonovalent. In one embodiment, such fragments can be combined with oneanother, or with other antibody fragments or receptor ligands to form“chimeric” binding molecules. Significantly, such chimeric molecules maycontain substituents capable of binding to different epitopes of thesame molecule, or they may be capable of binding to an activated proteinC epitope and a “non-activated protein C” epitope.

A monoclonal antibody can be readily prepared through use of well-knowntechniques such as those exemplified in U.S. Pat. No. 4,196,265, hereinincorporated by reference. Typically, a technique involves firstimmunizing a suitable animal with a selected antigen (e.g., apolypeptide or polynucleotide of the present invention) in a mannersufficient to provide an immune response. Rodents such as mice and ratsare preferred animals. Spleen cells from the immunized animal are thenfused with cells of an immortal myeloma cell. Where the immunized animalis a mouse, a preferred myeloma cell is a murine NS-1 myeloma cell.

The fused spleen/myeloma cells are cultured in a selective medium toselect fused spleen/myeloma cells from the parental cells. Fused cellsare separated from the mixture of non-fused parental cells, for example,by the addition of agents that block the de novo synthesis ofnucleotides in the tissue culture media. Exemplary and preferred agentsare aminopterin, methotrexate, and azaserine. Aminopterin andmethotrexate block de novo synthesis of both purines and pyrimidines,whereas azaserine blocks only purine synthesis. Where aminopterin ormethotrexate is used, the medium is supplemented with hypoxanthine andthymidine as a source of nucleotides. Where azaserine is used, themedium is supplemented with hypoxanthine.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants for reactivitywith antigen-polypeptides. The selected clones can then be propagatedindefinitely to provide the monoclonal antibody.

By way of specific example, to produce a monoclonal antibody, mice areinjected intraperitoneally with between about 1-200 μg of an antigencomprising a polypeptide. B lymphocytes are stimulated to grow byinjecting the antigen in association with an adjuvant such as completeFreund's adjuvant (a non-specific stimulator of the immune responsecontaining killed Mycobacterium tuberculosis). At some time (e.g., atleast two weeks) after the first injection, mice are boosted byinjection with a second dose of the antigen mixed with incompleteFreund's adjuvant.

A few weeks after the second injection, mice are tail bled and the seratitered by immunoprecipitation against radiolabeled antigen. Preferably,the process of boosting and titering is repeated until a suitable titeris achieved. The spleen of the mouse with the highest titer is removedand the spleen lymphocytes are obtained by homogenizing the spleen witha syringe. Typically, a spleen from an immunized mouse containsapproximately 5×10⁷ to 2×10⁸ lymphocytes.

Mutant lymphocyte cells known as myeloma cells are obtained fromlaboratory animals in which such cells have been induced to grow by avariety of well-known methods. Myeloma cells lack the salvage pathway ofnucleotide biosynthesis. Because myeloma cells are tumor cells, they canbe propagated indefinitely in tissue culture, and are thus denominatedimmortal. Numerous cultured cell lines of myeloma cells from mice andrats, such as murine NS-1 myeloma cells, have been established.

Myeloma cells are combined under conditions appropriate to foster fusionwith the normal antibody-producing cells from the spleen of the mouse orrat injected with the antigen/polypeptide. Fusion conditions include,for example, the presence of polyethylene glycol. The resulting fusedcells are hybridoma cells. Like myeloma cells, hybridoma cells growindefinitely in culture.

Hybridoma cells are separated from unfused myeloma cells by culturing ina selection medium such as HAT medium (hypoxanthine, aminopterin,thymidine). Unfused myeloma cells lack the enzymes necessary tosynthesize nucleotides from the salvage pathway because they are killedin the presence of aminopterin, methotrexate, or azaserine. Unfusedlymphocytes also do not continue to grow in tissue culture. Thus, onlycells that have successfully fused (hybridoma cells) can grow in theselection medium.

Each of the surviving hybridoma cells produces a single antibody. Thesecells are then screened for the production of the specific antibodyimmunoreactive with an antigen/polypeptide. Limiting dilution of thehybridomas isolates single cell hybridomas. The hybridomas are seriallydiluted many times and, after the dilutions are allowed to grow, thesupernatant is tested for the presence of the monoclonal antibody. Theclones producing that antibody are then cultured in large amounts toproduce an antibody in convenient quantity.

Liaw et al. (2003), incorporated herein by reference in its entirety,described preparations of certain mouse monoclonal antibodies againsthuman activated and human unactivated protein C.

Where the antibodies or their fragments are intended for therapeuticpurposes, it may desirable to “humanize” them in order to attenuate anyimmune reaction. Such humanized antibodies may be studied in an in vitroor an in vivo context. Humanized antibodies may be produced, for exampleby replacing an immunogenic portion of an antibody with a corresponding,but non-immunogenic portion (i.e., chimeric antibodies). Robinson etal., PCT Application PCT/U.S.86/02269; Akira et al., EP Application184,187; Taniguchi, EP Application 171,496; Morrison et al., EPApplication 173,494; Neuberger et al., PCT Application WO 86/01533;Cabilly et al., EP Application 125,023; Better et al. (1988); Liu et al.(1987); Liu et al. (1987); Sun et al. (1987); Nishimura et al. (1987);Wood et al. (1985); Shaw et al. (1988); all of which references areincorporated herein by reference. General reviews of “humanized”chimeric antibodies are provided by Morrison (1985) and by Oi et al.(1986); which references are incorporated herein by reference).

Suitable “humanized” antibodies can alternatively be produced by CDR orCEA substitution. Jones et al. (1986); Verhoeyan et al. (1988); Beidleret al. (1988); all of which references are incorporated herein byreference.

D. Pharmaceutical Compositions

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more antibodies, therapeutic agents oradditional agent dissolved or dispersed in a pharmaceutically acceptablecarrier. Aqueous compositions of the present invention comprise aneffective amount of the antibody, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. The phrases“pharmaceutically or pharmacologically acceptable” refer to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, or a human, asappropriate.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). The use of such media and agents for pharmaceuticalactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active ingredient,its use in the therapeutic compositions is contemplated. Supplementaryactive ingredients can also be incorporated into the compositions. Forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologic Standards.

The biological material should be extensively dialyzed to removeundesired small molecular weight molecules and/or lyophilized for moreready formulation into a desired vehicle, where appropriate. The activecompounds will then generally be formulated for parenteraladministration, e.g., formulated for injection via the intravenous,intramuscular, sub-cutaneous, intranasal, intralesional, or evenintraperitoneal routes. Typically, such compositions can be prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for using to prepare solutions or suspensions upon the additionof a liquid prior to injection can also be prepared; and thepreparations can also be emulsified.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The antibodies of the present invention can be formulated into acomposition in a free base, in a neutral or salt form. Pharmaceuticallyacceptable salts, include the acid addition salts (formed with the freeamino groups of the protein) and which are formed with inorganic acidssuch as, for example, hydrochloric or phosphoric acids, or such organicacids as acetic, oxalic, tartaric, mandelic, and the like. Salts formedwith the free carboxyl groups can also be derived from inorganic basessuch as, for example, sodium, potassium, ammonium, calcium, or ferrichydroxides, and such organic bases as isopropylamine, trimethylamine,histidine, procaine and the like.

The carrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The preparation of more, or highly, concentratedsolutions for direct injection is also contemplated, where the use ofDMSO as solvent is envisioned to result in extremely rapid penetration,delivering high concentrations of the active agents to a small area.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above,but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, intranasal, and intraperitonealadministration. In this connection, sterile aqueous media which can beemployed will be known to those of skill in the art in light of thepresent disclosure. For example, one dosage could be dissolved in 1 mlof isotonic NaCl solution and either added to 1000 ml of hypodermoclysisfluid or injected at the proposed site of infusion, (see for example,“Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and1570-1580). Some variation in dosage will necessarily occur depending onthe condition of the subject being treated. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual subject.

In addition to the compounds formulated for parenteral administration,such as intravenous or intramuscular injection, other pharmaceuticallyacceptable forms include, e.g., tablets or other solids for oraladministration; liposomal formulations; time release capsules; and anyother form currently used, including cremes.

In certain embodiments, the use of liposomes and/or nanoparticles iscontemplated for the formulation and administration of the antibodiesand/or analogs thereof. The formation and use of liposomes is generallyknown to those of skill in the art, and is also described below.

Nanocapsules can generally entrap compounds in a stable and reproducibleway. To avoid side effects due to intracellular polymeric overloading,such ultrafine particles (sized around 0.1 μm) should be designed usingpolymers able to be degraded in vivo. Biodegradablepolyalkyl-cyanoacrylate nanoparticles that meet these requirements arecontemplated for use in the present invention, and such particles areeasily made.

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200-500 Å, containing an aqueous solution in the core.

The following information may also be utilized in generating liposomalformulations. Phospholipids can form a variety of structures other thanliposomes when dispersed in water, depending on the molar ratio of lipidto water. At low ratios the liposome is the preferred structure. Thephysical characteristics of liposomes depend on pH, ionic strength andthe presence of divalent cations. Liposomes can show low permeability toionic and polar substances, but at elevated temperatures undergo a phasetransition which markedly alters their permeability. The phasetransition involves a change from a closely packed, ordered structure,known as the gel state, to a loosely packed, less-ordered structure,known as the fluid state. This occurs at a characteristicphase-transition temperature and results in an increase in permeabilityto ions, sugars and drugs.

Liposomes interact with cells via four different mechanisms: Endocytosisby phagocytic cells of the reticuloendothelial system such asmacrophages and neutrophils; adsorption to the cell surface, either bynonspecific weak hydrophobic or electrostatic forces, or by specificinteractions with cell-surface components; fusion with the plasma cellmembrane by insertion of the lipid bilayer of the liposome into theplasma membrane, with simultaneous release of liposomal contents intothe cytoplasm; and by transfer of liposomal lipids to cellular orsubcellular membranes, or vice versa, without any association of theliposome contents. Varying the liposome formulation can alter whichmechanism is operative, although more than one may operate at the sametime.

The therapeutic agent may comprise different types of carriers dependingon whether it is to be administered in solid, liquid or aerosol form,and whether it needs to be sterile for such routes of administration asinjection. The present invention can be administered intravenously,intradermally, intraarterially, intraperitoneally, intralesionally,intracranially, intraarticularly, intraprostaticaly, intrapleurally,intratracheally, intranasally, intravitreally, intravaginally,intrarectally, topically, intratumorally, intramuscularly,intraperitoneally, subcutaneously, subconjunctival, intravesicularlly,mucosally, intrapericardially, intraumbilically, intraocularally,orally, topically, locally, by inhalation (e.g., aerosol inhalation), byinjection, by infusion, by continuous infusion, localized perfusionbathing target cells directly, via a catheter, via a lavage, in cremes,in lipid compositions (e.g., liposomes), or by other methods or anycombination of the foregoing as would be known to one of ordinary skillin the art (see, for example, Remington's Pharmaceutical Sciences, 18thEd. Mack Printing Company, 1990, incorporated herein by reference).

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and the route of administration.The practitioner responsible for administration will, in any event,determine the concentration of active ingredient(s) in a composition andappropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, an active compound may comprise between about 2% to about75% of the weight of the unit, or between about 25% to about 60%, forexample, and any range derivable therein. In other non-limitingexamples, a dose may also comprise from about 1 microgram/kg/bodyweight, about 5 microgram/kg/body weight, about 10 microgram/kg/bodyweight, about 50 microgram/kg/body weight, about 100 microgram/kg/bodyweight, about 200 microgram/kg/body weight, about 350 microgram/kg/bodyweight, about 500 microgram/kg/body weight, about 1 milligram/kg/bodyweight, about 5 milligram/kg/body weight, about 10 milligram/kg/bodyweight, about 50 milligram/kg/body weight, about 100 milligram/kg/bodyweight, about 200 milligram/kg/body weight, about 350 milligram/kg/bodyweight, about 500 milligram/kg/body weight, to about 1000 mg/kg/bodyweight or more per administration, and any range derivable therein. Innon-limiting examples of a derivable range from the numbers listedherein, a range of about 5 mg/kg/body weight to about 100 mg/kg/bodyweight, about 5 microgram/kg/body weight to about 500 milligram/kg/bodyweight, etc., can be administered, based on the numbers described above.

In any case, the composition may comprise various antioxidants to retardoxidation of one or more component.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes)and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin; by the maintenanceof the required particle size by dispersion in carriers such as, forexample liquid polyol or lipids; by the use of surfactants such as, forexample hydroxypropylcellulose; or combinations thereof. In many cases,it will be preferable to include isotonic agents, such as, for example,sugars, sodium chloride or combinations thereof.

In other embodiments, one may use eye drops, nasal solutions or sprays,aerosols or inhalants in the present invention. Such compositions aregenerally designed to be compatible with the target tissue type. In anon-limiting example, nasal solutions are usually aqueous solutionsdesigned to be administered to the nasal passages in drops or sprays.Nasal solutions are prepared so that they are similar in many respectsto nasal secretions, so that normal ciliary action is maintained. Thus,in preferred embodiments the aqueous nasal solutions usually areisotonic or slightly buffered to maintain a pH of about 5.5 to about6.5. In addition, antimicrobial preservatives, similar to those used inophthalmic preparations, drugs, or appropriate drug stabilizers, ifrequired, may be included in the formulation. For example, variouscommercial nasal preparations are known and include drugs such asantibiotics or antihistamines.

In certain embodiments the antibodies are prepared for administration bysuch routes as oral ingestion. In these embodiments, the solidcomposition may comprise, for example, solutions, suspensions,emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatincapsules), sustained release formulations, buccal compositions, troches,elixirs, suspensions, syrups, wafers, or combinations thereof. Oralcompositions may be incorporated directly with the food of the diet.Preferred carriers for oral administration comprise inert diluents,assimilable edible carriers or combinations thereof. In other aspects ofthe invention, the oral composition may be prepared as a syrup orelixir. A syrup or elixir, may comprise, for example, at least oneactive agent, a sweetening agent, a preservative, a flavoring agent, adye, a preservative, or combinations thereof.

In certain preferred embodiments an oral composition may comprise one ormore binders, excipients, disintegration agents, lubricants, flavoringagents, and combinations thereof. In certain embodiments, a compositionmay comprise one or more of the following: a binder, such as, forexample, gum tragacanth, acacia, cornstarch, gelatin or combinationsthereof; an excipient, such as, for example, dicalcium phosphate,mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate or combinations thereof; a disintegratingagent, such as, for example, corn starch, potato starch, alginic acid orcombinations thereof; a lubricant, such as, for example, magnesiumstearate; a sweetening agent, such as, for example, sucrose, lactose,saccharin or combinations thereof; a flavoring agent, such as, forexample peppermint, oil of wintergreen, cherry flavoring, orangeflavoring, etc.; or combinations of the foregoing. When the dosage unitform is a capsule, it may contain, in addition to materials of the abovetype, carriers such as a liquid carrier. Various other materials may bepresent as coatings or to otherwise modify the physical form of thedosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both.

Additional formulations which are suitable for other modes ofadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum, vagina or urethra. After insertion, suppositoriessoften, melt or dissolve in the cavity fluids. In general, forsuppositories, traditional carriers may include, for example,polyalkylene glycols, triglycerides or combinations thereof. In certainembodiments, suppositories may be formed from mixtures containing, forexample, the active ingredient in the range of about 0.5% to about 10%,and preferably about 1% to about 2%.

The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminum monostearate,gelatin or combinations thereof.

E. Kits

Any of the compositions described herein may be comprised in a kit. Thekits will thus comprise, in suitable container means, an antibody and/oran additional agent of the present invention. The inventors envisageother components that may be included in a kit. Therapeutic kits of thepresent invention comprise in suitable container means, apharmaceutically acceptable formulation of an antibody in apharmaceutically acceptable formulation. The kit may have a singlecontainer means, and/or it may have distinct container means for eachcompound.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred. The antibody may also beformulated into a syringeable composition, in which case, the containermeans may itself be a syringe, pipette, and/or other such likeapparatus, from which the formulation may be applied to an infected areaof the body, injected into an animal, and/or even applied to and/ormixed with the other components of the kit.

However, the components of the kit may be provided as dried powder(s).When reagents and/or components are provided as a dry powder, the powdercan be reconstituted by the addition of a suitable solvent. It isenvisioned that the solvent may also be provided in another containermeans.

The container means will generally include at least one vial, test tube,flask, bottle, syringe and/or other container means, into which theantibody/antibody formulation is placed, preferably, suitably allocated.The kits may also comprise a second container means for containing asterile, pharmaceutically acceptable buffer and/or other diluent.

The kits of the present invention will also typically include a meansfor containing the vials in close confinement for commercial sale, suchas, e.g., injection and/or blow-molded plastic containers into which thedesired vials are retained.

Irrespective of the number and/or type of containers, the kits of theinvention may also comprise, and/or be packaged with, an instrument forassisting with the injection/administration and/or placement of theultimate antibody within the body of an animal. Such an instrument maybe a syringe, pipette, forceps, and/or any such medically approveddelivery vehicle.

F. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE 1 Methods for Screening, Identification and Use of a Human MAbof the Present Invention

Materials. Human protein C, bovine thrombin were prepared as describedpreviously (Esmon et al., 1993; incorporated herein by reference in itsentirety). Recombinant APC (Xigris) was from Eli Lilly. Spectroxyme PCawas from American Diagnostica. 1 -Palmitoyl-2-oleoyl-phosphatidylcholine(PC), 1-palmitoyl-2-oleolylphosphatidylserine (PS), and1-palmitoyl-2-oleolyl-phosphatidylethanolamine (PE) were from AvantiPolar Lipids, Inc. Human endothelial derived EA.hy926 cells weremaintained in DMEM (Dulbecco's modified Eagle's medium) supplementedwith 10% fetal bovine serum, L-glutamine and HAT (hypoxanthine,aminopterin, thymidine). Fluorescein labeled APC (FL-APC) was preparedwith the Fluorescein-EX Protein Labeling Kit from Molecular Probesaccording to the manufacturer's instructions. In these examples,“protein C,” without the “activated” modifier, refers to unactivatedprotiein C.

Generation of mouse anti-human protein C and APC monoclonal antibodies.Mouse monoclonal antibody (mAb) against human protein C or APC wasdeveloped by standard techniques (Rezaie and Esmon, 1992).

Screening for specific mAb for protein C or APC. Human protein C mAb1575 and 1580 (HPC1575 and HPC1580) were obtained by screening theblocking ability of mAb for the binding of FL-APC to EA.hy926 cells byFACS. Briefly, EA.hy926 cells were incubated with 50 nM FL-protein C and100 nM various monoclonal antibodies against protein C in HBSS (Hanks'Balanced Salt Solution) buffer containing 0.5% BSA, 3 mM CaCl₂ and 0.6mM MgCl₂ for 30 min on ice, and subjected to FACS analysis. Human APCmAb 1573 (HAPC1573) was obtained by screening the binding ability of mAbto APC but not protein C with an ELISA assay. Briefly, a 96-wellMaxiSorp plate (NUNC) was coated with 5 μg/ml different mAbs in 15 mMNa₂CO₃, 35 mM NaHCO3,pH 9.6 buffer overnight at 4° C. The plate waswashed with TTBS (TBS containing 0.05% Tween-20) containing 1 mM CaC12(TTBS calcium buffer), blocked with 0.1% gelatin in TBS (20 mM Tris-HCl,150 mM NaCl, pH 7.5) for 1 hr, washed with TTBS calcium buffer again,incubated with 100 ng/ml protein C or APC in TTBS calcium buffer for 1hr. After washing with TTBS calcium buffer, the plate was incubated with2 μg/ml biotinylated HPC1580 for 1 hr, washed again with TTBS calciumbuffer, incubated with 1 μg/ml streptavidin-alkaline phosphataseconjugate in TTBS calcium buffer for another hour. The endpointabsorbance at 405 nm was read on a Vmax microplate reader after finalwashing with TTBS calcium buffer and adding p-nitrophenyl phosphateliquid substrate (Sigma).

ELISA assay for APC in plasma. The assay was modified from thepreviously described ELISA assay for screening mAb against APC. Briefly,the plate was coated with 5 μg/ml HAPC1573, blocked with TBS containing1× casein (Vector Lab) and washed again with TTBS calcium buffer. Spikerecombinant APC from 0-8 ng/ml in TTBS containing 10 mM benzamidine, 1mM EDAT and 0.25× casein buffer (Dilution buffer) or 1:4 diluted humanplasma and incubate the samples in the plate for one hour. After washingwith TTBS calcium buffer, the plate was incubated with 1 ug/mlbiotinylated HPC1575 in TTBS containing 10 mM benzamidine, 5 mM CaCl2and 0.25× casein buffer for one hr. After washing with TTBS calciumbuffer, the plate was incubated with 0.5 μg/ml streptavidin-HRP in TTBScontaining 10 mM benzamidine, 5 mM CaCl₂ and 0.25× casein buffer foranother hour, washed again with TTBS calcium buffer, and color developedwith Ultra-TMB substrate (Pierce). OD450 was read after adding 0.5 MH2SO4 to stop the HRP enzymatic reaction.

Influence of mAb on FL-APC binding on endothelium. EA.hy926 cells wereincubated with 50 nM FL-APC in HBSS buffer containing 0.5% BSA, 3 mMCaCl2 and 0.6 mM MgCl2 in the absence or presence of HAPC1573 or HPC1575in various concentrations for 30 min on ice, and subjected to FACSanalysis.

Influence of mAb on APC anticoagulant activity in plasma clotting assay.The influence of mAb on APC anticoagulant activity in plasma wasdetermined in a modified factor Xa one-stage clotting assay using ST4coagulation instrument (Diagnostica Stago). In the standard assay, humannormal pool plasma was added adjusted amount of X-CP, a factorX-activating enzyme from Russell's viper venom, to give a 30-s clottingtime in the mixture of phospholid vesicles (final 10 μg/ml of 40% PE,20% PS and 40% PC, w/v) and CaCl₂ (6.25 mM) in TBS containing 0.1% BSA.Clotting was initiated with CaCl₂ addition. APC (final 200 ng/ml) orHAPC1573 (final 20 μg/ml) was added before CaCl₂ addition.

Influence of mAb on APC amidolytic activity toward a chromogenicsubstrate. The amidolytic activity of 50 μl of 10 nM APC in HBSS buffer(HBSS containing 0.1% bovine serum albumin, 3 mM CaCl₂, 0.6 mM MgCl₂) inthe absence or presence of 66.7 nM HPC1555 or HAPC1573 was determinedwith adding 50 μl of 0-2 mM serial diluted Spectrozyme PCa in 50 mMHEPES, 100 mM NaCl, pH 7.5 buffer.

Histone cytotoxicity assay. EA.hy926 cells were incubated with calfthymus histone H3 or H4 (Roche) in the absence or presence of 100 nM APCand 200 nM HAPC1573 in Opti-MEM medium (Invitrogen) for 1 hr at 37° C.and then 5 min at RT after 10 μg/ml propidium iodide (PI) was added.Cells were washed and detached with EDTA/PBS and subjected to flowcytometry for PI positive staining.

EXAMPLE 2 Results for Screening, Identification and Use of a Human MAbof the Present Invention

HAPC1573 enhances APC binding on endothelium. To test whether HAPC1575would have any influence on APC binding on endothelium, the inventorsincubated EA.hy926 cells with FL-APC in the absence or presence ofHAPC1573 or HPC 1575 and measured the binding of FL-APC on cells by flowcytometry. The histograph of flow cytometry showed that HAPC1573enhanced FL-APC binding on the endothelial cells, while HPC1575inhibited the binding of FL-APC on the cells (FIG. 2). HAPC 1573facilitates APC internalization on endothelium FL-APC could beinternalized into EA.hy926 cells through the interaction of Gla domainof APC and EPCR on the cells, and this internalization could be blockedby either EPCR blocking Ab (JRK1494) or Gla domain blocking Ab (HPC1575)(FIG. 3). HAPC1573 could facilitated FL-APC internalization into thecells, and this effect could be completely blocked by EPCR blocking Ab(FIG. 3).

HAPC1573 alters APC amidolytic activity toward a chromogenic substrate.Since HAPC1573 recognized APC on the ELISA plate and on endothelialcell, the inventorsasked if HAPC1573 could affect the amidolyticactivity of APC for chromogenic substrate. Synthetic peptide substratesare usually small molecules, about a few hundred Dalton molecularweight, most antibodies against serine proteases in plasma have littleeffect on the enzymatic activity for these small substrates. However,HAPC1573 dramatically altered the kinetic parameters of APC toward itschromogenic substrate, Spectrozyme PCa (FIG. 4). The km of APC towardSpectrozyme PCa was 15 nM in the presence of HAPC1573, compared to 270nM in the absence of Ab or presence of HPC1555. The kcat of APC towardSpectrozyme PCa was 18 in the presence of HAPC1573, compared to 67 inthe absence of Ab or in the presence of HPC1555. This profound change ofAPC toward small peptide substrate in the presence of HAPC1573 indicatedthat this mAb recognized an epitope near active site of APC and theinteraction of Ab and antigen dramatically increased the affinity of APCtoward small peptide substrate but decreased the off rate of productfrom APC catalytic site.

HAPC1573 blocks APC anticoagulant activity in plasma. FIG. 5 showed thatHAPC1573 almost completely diminished the prolongation effect of APC infactor Xa initiated one-stage plasma clotting assay, suggesting that theinteraction of HAPC1573 and APC prevents APC from cleaving factor Va.

The influence of HAPC1573 on APC cleaving extracellular histones.Recently, the inventors found that APC could cleave extracellularhistones and protect endothelium from cytotoxicity of histones(manuscripts in preparation). Since HAPC1573 altered APC amidolyticactivity toward the chromogenic substrate and blocked APC anticoagulantactivity in plasma, the inventors asked if this mAb could affect APCcleaving extracellular histone H3 and H4, and affect APC cytoprotectionon endothelium against histone H3 and H4 cytotoxicity.

Surprisingly, HAPC1573 did not inhibit but actually enhanced APCcleaving histone H3 and H4 (FIG. 6). Consistently, HAPC1573 did notinhibit but slightly enhanced APC cytoprotection activity on endotheliumagainst histone H3 and H4 (FIG. 7).

EXAMPLE 3 Discussion for Screening, Identification and Use of a HumanMAb of the Present Invention

Protein C is activated by thrombin complexed with thrombomodulin onendothelium. Unlike the few-second transient life of active thrombin invivo, human APC has about a 20 minute half-life in circulation after itsgeneration (Berg, et al., 2003). Therefore, one may feasibly measure alevel of APC in plasma to study its regulation under variouspathophysical conditions.

The currently available methods to measure APC are based on an enzymecapture assay, which uses an antibody capturing APC and then measuresAPC activity by chromogenic substrates. Since all antibodies used inthese assays recognized not only APC but also its zymogen, protein C,and since protein C concentration is about 1000 times more than APC innormal circulation, APC measurement using these methods is notclinically relevant. A rapid and robust method for APC measurement isdesirable in both diagnosis and treatment. The results above show that amAb, HAPC1573, recognizes APC but not protein C and demonstrates thedevelopment of a convenient ELISA method for measuring APC level inplasma in vivo. Typically, it takes less than 4 hours to measure aplasma sample containing 1 ng/ml APC with this method compared to 19hours or even weeks with enzyme capture assays (Gruber and Griffen,1992; Liaw et al., 2003).

Recent studies have shown that anticoagulant activity of APC isdispensable for its cytoprotective function, but APC cleavage activitytoward PAR1 might be essential for its anti-apoptotic effect (Mosnier etal., 2004). However, the cytoprotection effect of APC has been shown notonly in endothelial cells which express EPCR, but also on other cellssuch as neuron and keratinocytes which do not express EPCR on their cellsurfaces (Guo et al., 2004; Berg et al., 2003), indicating othermechanisms than PAR1 mediated APC signaling might exist. HAPC1573altered APC cleavage activity toward a chromogenic peptide substrate andalso blocked APC anticoagulant activity in a plasma clotting assay,suggesting this mAb recognizes an epitope near the APC active site andalters its catalytic activity upon antibody-antigen binding.

On the other hand, HAPC1573 did not inhibit but actually enhanced APCcleaving extracellular histones, and enhanced APC cytoprotectionactivity on endothelium against histones, indicating that APCanticoagulant activity for cleaving activated factor V and VIII is notrequired for its cytoprotection activity by cleaving extracellularhistones. Cleaving extracellular histones independent from itsanticoagulant activity might be one of the molecular mechanisms of APCregulation inflammation and cytoprotection.

HAPC1573 may, for example, be used in treatment of hemophilia Apatients. APC cleaves both factor Villa and factor Va and thusnegatively regulates blood clotting. In hemophilia A patients, factorVIII levels are low and the inactivation of factor Va by APC is probablya major pathway to regulate hemostasis and thrombosis in these patients.Recent clinical reports demonstrated factor V Laiden mutant which isresistant to APC cleavage was beneficial to hemophilia A patientsregarding their bleeding symptom (van't Zant et al., 1997). Blocking APCanticoagulant activity toward factor Va in vivo by a mAb such asHAPC1573 might be an alternative approach for hemophilia A treatments,especially for those patients who have high level factor VIII inhibitorsso that the factor VIII replacement therapy would not be very effective.

Another possible clinical application of HAPC1573 is in the treatment oftrauma patients wherein homeostasis is disrupted, excessive bleeding islikely, and surgical intervention is delayed to regain homeostatis.Treatment with HAPC1573 can selectively restore the pro-coagulant statewithout eliminating the cytoprotective or anti-inflammatory activitiesof activated protein C.

Another possible clinical application of HAPC1573 is its combinationwith APC in sepsis treatment. APC is currently the only FDA approvedmedication for severe sepsis. Its bleeding side effect in patients isdue to APC anticoagulant activity. HAPC1573 blocked APC anticoagulantactivity while maintained and even enhanced APC cytoprotective effect.This mAb-APC complex might be a better therapeutic than APC aloneregarding its bleeding side effect.

EXAMPLE 4 Methods for Screening and Use of a Mouse Monoclonal Antibodyof the Present Invention

Materials and Methods. Recombinant mouse protein C, APC, rat mAb MPC1609and MAPC1591 were produced in this lab according to the standardprocedures. Fluorescein labeled APC (FL-APC) was prepared with theFluorescein-EX Protein Labeling Kit from Molecular Probes according tothe manufacturer's instructions.

Animal study. Six to 12 week male BL6 mice were used in this studyaccording to the animal protocol approved by Institutional Animal Careand Use Committees of the Oklahoma Medical Research Foundation.

Cell culture. bEnd3 cells (mouse brain derived endothelial cell line)were cultured in DMEM (Dulbecco's modified Eagle's medium) supplementedwith 10% fetal bovine serum and L-glutamine. EA.hy926 cells (humanendothelial cell line) were cultured in DMEM supplemented with 10% fetalbovine serum, L-glutamine and HAT (hypoxanthine, aminopterin,thymidine).

FL-APC binding and protein C activation on endothelium. bEnd3 cells wereincubated with 100 nM FL-APC in HBSS buffer containing 0.5% BSA, 3 mMCaCl₂ and 0.6 mM MgCl₂ in the absence or presence of 125 nM MPC1609 orMAPC1591 for 15 minute on ice, and subjected to FACS analysis.

The influence of mAb on protein C activation on endothelial cells. bEnd3cells in 24-well plate were washed once with HBSS buffer (HBSScontaining 0.1% bovine serum albumin, 3 mM CaCl₂, 0.6 mM MgCl2) andpreincubated for 5 min with HBSS buffer containing 0.1 μM protein C with0.1 μM MPC1609 or MAPC1591. The activation reactions were initiated byaddition of 5 nM bovine thrombin in a total volume of 0.2 ml. After 15min at 37° C., the reactions were stopped by adding 50 μl of bovineanti-thrombin III (1.67 mg/ml) to the reactions. Fifty μl supernatantswere transferred to the 96-well microplate, and the activation rate ofprotein C were determined with Vmax at 405 nm toward 50 μl of 0.4 mMSpectrozyme PCa substrate in 100 mM NaCl, 50 mM HEPES-NaOH, pH 7.5buffer.

APC anti-coagulant activity assay. The influence of mAb on APCanti-coagulant activity in plasma was determined in a modified factor Xaone-stage clotting assay using ST4 coagulation instrument (DiagnosticaStago). In this assay, plasma (50% mouse plasma and 50% human normalpool plasma) was added adjusted amount of X-CP, a factor X-activatingenzyme from Russell's viper venom, to give a 30-s clotting time in themixture of phospholipid vesicles (final 10 μg/ml of 40%phosphatidylethonalamine, 20% phosphatidylserine and 40%phosphatidylcholine, w/v) and CaCl₂ (6.35 nM) in 20 mM Tris-HCl, 150 mMNaCl buffer (pH 7.5) containing 0.1% BSA. Clotting was initiated withCaCl₂ addition. APC (final 200 ng/ml) and MPC1609 (final 5 μg/m1) orMAPC1591 (final 5 μg/ml) were added before CaCl₂ addition.

Histone cytotoxicity assay. EA.hy926 cells were incubated with 50 μg/mlcalf thymus histones (Sigma) in the absence or presence of 100 nM APCand 200 nM MAPC1591 in Opti-MEM medium (Invitrogen) for 1 hr at 37° C.and then 5 min at RT after 10 μg/m1 propidium iodide (PI) was added.Cells were washed and detached with 0.526 mM EDTA in PBS and subjectedto flow cytometry for PI-positive staining.

IL-6, BUN and creatinine assay. Mouse serum was analyzed on Vitros 250Chemistry Analyzer (Ortho-Clinical Diagnostics) for BUN and creatinine.Serum IL-6 was measured by Quantikine Colorimetric Sandwich ELISA (R&DSystems).

EXAMPLE 5 Results for Screening and Use of a Mouse Monoclonal Antibodyof the Present Invention

MPC1609 was against both protein C and APC, and could inhibit protein Cand APC binding on endothelium (FIG. 8A and data not shown). MAPC1591was against APC but not protein C and could enhance APC binding onendothelium (FIG. 8A and data not shown). Protein C activation onendothelium was dramatically decreased in the presence of MPC1609 (FIG.8B). MAPC1591 also decreased protein C activation to some extentprobably due to its enhancement of APC binding on the cells (FIG. 8B).Both MPC1609 and MAPC1591 could completely inhibit APC anti-coagulantactivity in plasma clotting assay (FIG. 8C). Based on these in vitrostudies, the inventors concluded that MPC1609 inhibited protein C andAPC binding on endothelium or phospholipid by masking the Gla-domain ofprotein C or APC which was responsible for the binding of protein C orAPC on endothelium or phospholipid. MAPC1591 recognized APC but notprotein C through interacting with an epitope around the active site ofAPC and this interaction inhibited APC anti-coagulant activity likely bypreventing APC cleaving factor Va.

To explore the molecular mechanism of APC protective effect in LPSinduced septic shock, the inventors injected sublethal dose of LPS withMPC1609 or MAPC1591 into mice and they found that mice injected LPS andMPC1609 all died within 48 hrs, mice injected LPS and MAPC1591 allsurvived (FIG. 9). 18 hour after infusion, mice injected with LPS andMPC1609 showed severe septic shock symptoms including low bodytemperature, high neutrophil, low lymphocyte and low platelet counts inperipheral blood (FIG. 10A and data not shown). Serum IL-6 level wasextremely higher in these mice (FIG. 10B), but no significant differenceof IL-6 mRNA level in heart, lung, liver, spleen, thymus and peripheralblood was observed between these mice and mice injected with LPS plusMAPC1591 (data not shown), suggesting that de novo synthesis of IL-6might not be a major cause for the sustained higher IL-6 level in theseptic shocked mice. Serum BUN and creatinine levels in mice injectedwith LPS and MPC1609 were higher than other mice (FIG. 10C) indicatedacute renal failure occurred in these mice which might contribute a slowclearance of IL-6. Interestingly, BUN and creatinine levels in miceinjected with LPS and MAPC1591 were even lower than the levels in miceinjected with LPS alone (FIG. 10C), suggesting that MAPC1591 and APCcomplex might be more effective than APC for the renal protection invivo under LPS challenge.

Extracellular histones were found on activated neutrophil andmacrophages, and apoptotic cells (Brinkmann et al., 2004; Radic et al.,2004), and they were cytotoxic toward mammalial cells (Abakushin et al.,1999; Currie et al., 1997; Kleine et al., 1997). APC could cleavehistone H3 and histone H4, and MAPC1591 did not inhibit but actuallyenhanced APC cleaving these histones (FIG. 10A). APC could reduce thecytotoxicity of histones toward endothelium and MAPC1591 could enhancethis APC cytoprotective activity (FIG. 11B). Extracellular histone wasindeed detected in the septic mouse serum after LPS and MPC1609injection but not in the mice injected with LPS or LPS and MAPC1591(FIG. 11C), indicating an in vivo correlation between the deficientprotein C activation, presence of extracellular histone in thecirculation and lethality of mice under septic shock. Taking these invitro data together with in vivo observations, the inventors coulddistinguish the cytoprotective activity of APC from its anticoagulantactivity by MAPC 1591, and cleaving cytotoxic histones by APCindependent of its anticoagulant activity provides a new molecularmechanism of APC preventing mice from LPS induced septic shock.

EXAMPLE 6 Discussion for Screening and Use of a Mouse MonoclonalAntibody of the Present Invention

The protein C pathway plays an important role in regulating both bloodcoagulation and inflammation (Esmon, 2006). Human APC was demonstratedto significantly reduce mortality in severe sepsis and has been approvedas the first medication for severe sepsis treatment (Bernard et al.,2001). However, the molecular mechanism of APC protective effects insepsis is still poorly understood. Mutagenesis study indicated thatanticoagulant activity of APC was apparently dispensable for APCanti-apoptotic effect on endothelial cells (Mosnier et al., 2004), andanti-inflammation and anti-apoptotic effects of APC signaling wereprotease activated receptor 1 (PAR-1) mediated in endothelial cells(Reiwald et al., 2002). However, PAR-1-deficient mice had similarphenotype to its wild-type control mice under LPS challenge, suggestingthat PAR-1 might not be a major player for APC to regulate inflammationand cytoprotection in vivo (Pawlinski et al., 2004; Camerer 2006). Givena central role of APC in regulating pathophysical functions in vivo, theinventors generated two mAbs against mouse protein C and mouse APC andused these two mAbs to explore the poorly understood mechanisms of APCprotective effect in LPS induced septic shock in mice.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

G. References

References mentioned throughout this application, including thefollowing references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference:

U.S. Pat. No. 4,196,265

Abakushin et al., Biochemistry (Most.), 64:693-698, 1999. Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring HarborPress, Cold Spring Harbor, N.Y., 1988. Beidler et al., J. Immunol.,141:4053-4060, 1988.

Berg et al., Proc. Natl. Acad. Sci. USA, 100:4423-4428, 2003.

Bernard et al., New England J. Medicine, 344:699-709, 2001. Better etal., Science, 240:1041-1043, 1988. Brinkmann, Science, 203:1532-1535,2004. Camerer, Blood, 107:3912-3921, 2006.

Currie et al., Biochim. Biophys. Acta, 1355:248-258, 1997.

EP Appin. 125,023 EP Appin. 171,496 EP Appin. 173,494 EP Appin. 184,187

Esmon, In: Natural anticoagulants and their pathways, Handbook of Exper.Pharm., 132:447-476, Born et al. (Eds.), Springer-Verlag, NY, 1999.Esmon, J. Biol. Chem., 264; 4743- 4746, 1989.

Esmon, Proteolytic Enzymes in Coagulation, Fibrinolysis, and ComplementActivation, Part A, 222:359-385, 1993.

Esmon, Semin. Thromb. Hemost., 32 Suppl 1:49-60, 2006.

Gruber and Griffin, Blood, 79:2340-2348, 1992. Guo et al., Neuron.,41:563-572, 2004. Jones et al., Nature, 321:552-525, 1986.

Kleine et al., Am. Physiol 273:C1925-C1936, 1997.

Liaw et al., J. Thrombosis and Haemostasis, 1:662-670, 2003. Liu et al.,J. Immunol., 139:3521-3526, 1987.

Liu et al., Proc. Natl. Acad. Sci. USA, 84:3439-3443, 1987.

Morrison, Science, 229:1202-1207, 1985. Mosnier et al., Blood,104:1740-1744, 2004.

Nishimura et al., Canc. Res., 47:999-1005, 1987.

Oi et al., BioTechniques, 4:214, 1986. Pawlinski et al., Blood,103:1342-1347, 2004.

PCT Appin. PCT/U.S.86/02269PCT Appin. WO 86/01533

Radic et al., J. Immunol., 172:6692-6700, 2004. Remington'sPharmaceutical Sciences, 18^(th) Ed., Mack Printing Company, 1289-1329,1990. Remington's Pharmaceutical Sciences, 15^(th) Ed., 1035-1038 and1570-1580, 1990.

Rezaie and Esmon, J. Biol. Chem., 267:26104-26109, 1992.

Riewald et al., Science, 296:1880-1882, 2002.

Shaw et al., J. Natl. Cancer Inst., 80:1553-1559, 1988.Sun et al., Proc. Natl. Acad. Sci. USA, 84:214-218, 1987.van't Zant et al., Blood, 90(8):3067-3072, 1997.

Verhoeyan et al., Science, 239:1534, 1988.

Wood et al., Nature, 314:446-449, 1985.

1-10. (canceled)
 11. A monoclonal antibody, wherein said antibodyinhibits activated or unactivated protein C binding of endothelial cellprotein C receptor (EPCR) or phospholipids and inhibits unactivatedprotein C activation. 12-15. (canceled)
 16. The antibody or of claim 11,wherein the antibody is a human antibody.
 17. The antibody of claim 11,wherein the antibody is a humanized antibody.
 18. The antibody of claim11, wherein the antibody is an antibody fragment.
 19. (canceled)
 20. Apharmaceutical composition comprising a monoclonal antibody, whereinsaid antibody (a) binds to activated protein C and inhibitsanticoagulant activity but does not bind to or inhibit activation ofunactivated protein C, or (b) inhibits activated or unactivated proteinC binding of endothelial cell protein C receptor (EPCR) or phospholipidsand inhibits unactivated protein C activation, and a pharmaceuticallyacceptable carrier.
 21. (canceled)
 22. A method of inhibiting activatedprotein C anticoagulant activity in a subject, comprising administeringan effective amount of a monoclonal antibody wherein said antibody bindsto activated protein C and inhibits anticoagulant activity but does notbind to or inhibit activation of unactivated protein C, to said subject.23. The method of claim 22, wherein the cytoprotective effects ofactivated protein C are not decreased by said monoclonal antibody.
 24. Amethod of inhibiting activated protein C amidolytic activity in asubject comprising administering an effective amount of a monoclonalantibody, wherein said antibody binds to activated protein C andinhibits anticoagulant activity but does not bind to or inhibitactivation of unactivated protein C, to said subject.
 25. A method oftreating a subject in need of blood coagulation comprising administeringan effective amount of a monoclonal antibody, wherein said antibodybinds to activated protein C and inhibits anticoagulant activity butdoes not bind to or inhibit activation of unactivated protein C, to saidsubject.
 26. A method of treating a subject in need of blood coagulationcomprising administering an effective amount of an antibody of claim 11to said subject.
 27. A method of treating a subject suffering fromsepsis comprising administering an effective amount of a monoclonalantibody, wherein said antibody binds to activated protein C andinhibits anticoagulant activity but does not bind to or inhibitactivation of unactivated protein C, to said subject.
 28. The method ofclaim 27, further comprising administration of activated protein C. 29.A method of treating a subject suffering from sepsis comprisingadministering an effective amount of an antibody of claim 11 to saidsubject.
 30. The method of claim 29, further comprising administrationof activated protein C.
 31. A method of treating a subject sufferingfrom hemophilia comprising administering an effective amount of amonoclonal antibody, wherein said antibody binds to activated protein Cand inhibits anticoagulant activity but does not bind to or inhibitactivation of unactivated protein C, to said subject.
 32. A method oftreating a subject suffering from hemophilia comprising administering aneffective amount of an antibody of claim 11 to said subject.
 33. Amethod of modulating hemostasis in a subject, comprising administeringan effective amount of a monoclonal antibody, wherein said antibody (a)binds to activated protein C and inhibits anticoagulant activity butdoes not bind to or inhibit activation of unactivated protein C, to saidsubject, or (b) inhibits activated or unactivated protein C binding ofendothelial cell protein C receptor (EPCR) or phospholipids and inhibitsunactivated protein C activation.
 34. The method of claim 33, whereinthe subject is a trauma patient. 35-36. (canceled)
 37. A method ofmodulating thrombosis in a subject, comprising administering aneffective amount of a monoclonal antibody, wherein said (a) antibodybinds to activated protein C and inhibits anticoagulant activity butdoes not bind to or inhibit activation of unactivated protein C, to saidsubject, or (b) inhibits activated or unactivated protein C binding ofendothelial cell protein C receptor (EPCR) or phospholipids and inhibitsunactivated protein C activation.
 38. (canceled)
 39. A method ofinhibiting activation of unactivated protein C activation comprisingadministering to a subject an effective amount of the monoclonalantibody of claim 11.